Isotopically-labeled proteome standards

ABSTRACT

The invention provides methods for quantifying biomolecules, such as polypeptides in mass spectrometric analysis. The methods include use of a biomolecule standard having at least one atomic isotope different than that of the naturally occurring isotopes in the biomolecule of interest. Methods of the present invention also include methods for quantifying biomolecules where the copy biomolecule standard is made by expressing the biomolecule using a recombinant cell. Further included are the biomolecule standards themselves, method for making such standards, kits, systems, reagents, and engineered cells relating to the use of biomolecule standards in mass spectrometric analysis.

RELATED APPLICATION

This patent application is a continuation and claims the right ofpriority under 35 U.S.C. §120 to U.S. patent application Ser. No.13/092,593 entitled “Isotopically-Labeled Proteome Standards”, filedApr. 22, 2011, now abandoned, is a divisional application and claims theright of priority under 35 U.S.C. §121 to U.S. patent application Ser.No. 11/368,996 entitled “Isotopically-Labeled Proteome Standards”, filedMar. 7, 2006, now U.S. Pat. No. 7,939,331, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 60/659,635 filed on7 Mar. 2005, entitled “Isotopically-Labeled Proteome Standards.” Theaforementioned patent applications are commonly owned with the presentapplication, and the contents thereof are hereby expressly incorporatedby reference in their entireties, including all text and figures.

FIELD OF THE INVENTION

The present invention relates generally to the field of massspectrometry analysis of biomolecules, and more specifically to massspectrometry analysis of proteins. The present invention providesisotopically-labeled biomolecule standards, such as protein standards,that may be used in mass spectrometry methods of quantifying abiomolecule in a sample. The present invention further provides methodsof making such biomolecule standards, reagents, and kits.

BACKGROUND

Mass spectrometry (MS) is a powerful analytical technique that is usedto identify unknown compounds, to relatively quantify known compounds,and to elucidate the structure and chemical properties of molecules.

Traditionally, proteome analysis has been performed using a combinationof high resolution gel electrophoresis, in particular, two-dimensional(2D) gel electrophoresis, to separate proteins, and mass spectrometry toidentify proteins. Typically, separation is by isoelectric focusing,which separates proteins by charge in a pH gradient, followed bySDS-PAGE, which separates proteins by molecular weight. After stainingand separation, the mixture appears as a two-dimensional array of spotsof separated proteins. Spots are excised from the gel, enzymaticallydigested, and subjected to mass spectrometry for identification.Relative quantitation of the identified proteins can be performed byobserving the relative intensities of the spots via image analysis ofthe stained gel. However, because ionization efficiency of differentprotein fragments varies greatly, comparative quantification using massspectrometry is unreliable.

Alternatively, peptides have been labeled isotopically before gelseparation and expression levels quantified by mass spectrometry orradiographic methods. Absolute concentrations have not been achievableusing these methods.

2D gels have a number of drawbacks. In particular, the approach issequential and tedious, and is additionally fundamentally limited inthat many biologically important classes of proteins, such as nuclearproteins or membrane proteins, are practically undetectable using thesemethods. Very acidic or basic proteins, very large or small proteins,and membrane proteins are either excluded or underrepresented in 2D gelpatterns. Low abundance proteins, including regulatory proteins, arerarely detected when entire cell lysates are analyzed, reflecting alimited dynamic range. These deficiencies are detrimental forquantitative proteomics.

Protein standards for 2D gels are generally known. 2D gels separateproteins and polypeptides based on size. For example, US Patentapplication 20030157720 discloses a protein standard set that is basedupon commercial protein standards providing polypeptides as standardsfor calculating molecular weight and the amount of peptide present.

However, in mass spectrometry, separation is not based on size alone,but separation is based on a mass to charge ratio. The mass to chargeratio is accurately obtained by MS. However, MS fragments are not easilyassigned to a particular protein without sequencing (which is possiblewith many popular MS machines). Different peptides have differentionization efficiencies and patterns for MS, which prevents accuratecomparison of one protein or polypeptide to another as is seen with 2Dgels. An ideal MS standard should behave identically to the proteinbeing measured during preparation for MS as well as within the MS.

Because it can provide detailed structural information, massspectrometry is currently a valued analytical tool for biochemicalmixture analysis and protein identification. For example, capillaryliquid chromatography combined with electrospray ionization tandem massspectrometry has been used for large-scale protein identificationwithout gel electrophoresis. Qualitative differences between spectra canbe identified, and proteins corresponding to peaks occurring in onlysome of the spectra are considered as candidate biological markers. Massspectrometry analyses are not quantitative, however. In most cases,quantitation in mass spectrometry requires an internal standard, acompound introduced into a sample at known concentration. Spectral peakscorresponding to sample components are compared with the internalstandard peak height or area for quantitation. Ideally an internalstandard has elution and ionization characteristics similar to andpreferably identical to those of the target compound but preferably thestandard generates ions with a detectably different mass-to-chargeratio.

Using internal standards for complex biological mixtures is problematic.In many cases, the compounds of interest are unknown a priori,preventing appropriate internal standards from being devised. Theproblem is more difficult when there are many compounds of interest. Inaddition, biological samples are often available in very low volumes,and addition of an internal standard can dilute mixture componentssignificantly. Low-abundance components, often the most relevant orsignificant ones, may be diluted to below noise levels and henceundetectable. Also, it can be difficult to judge the proper amount ofinternal standard to use. Thus internal standards are not widespreadsolutions to the problem of protein quantitation.

To reliably identify a biomolecule, such as a protein, from a sample,mass spectrometry (MS) based methods for proteomics rely onidentification of a fragment ion, for example a peptide generated by thesequence specific fragmentation of a protein. Therefore, biomolecules,such as proteins and carbohydrates generally need to be enzymatically orchemically fragmented prior to mass spectrometric analysis. A proteingenerally generates a large number of peptides and hence a large numberof peptides must be sequenced for each experiment.

Stable isotope labeling with amino acids in cell culture (SILAC) wasdeveloped as a useful tool for assaying relative concentrations ofproteins of cells grown in culture. SILAC incorporates a label intoproteins for mass spectrometric (MS)-based proteomics. SILAC relies onmetabolic incorporation of a “light” or “heavy” form of an amino acidinto proteins.

In a SILAC experiment, two groups of cells are grown in culture mediathat are essentially identical except in one respect: one media containsa “light” and the other a “heavy” form of a particular amino acid (fore.g. L-leucine or deuterated L-leucine). Thus, conventional SILACtechniques rely on growing parallel cultures where one set of culturesis grown in media containing an isotopically-labeled amino acid (such as¹⁵N-Arg) and the other culture set is grown in conventional mediathereby allowing an investigator to challenge one set of cultures withan external stimulus to monitor the relative changes in expression. Witheach cell doubling the cell population replaces at least half of theoriginal form of the amino acid, eventually incorporating 100% of agiven “light” or “heavy” form of the amino acid. Thus, when the labeledanalog of an amino acid is supplied to cells in culture instead of thenatural amino acid, it is incorporated into all newly synthesizedproteins. After a number of cell divisions, each instance of thisparticular amino acid will be replaced by its isotope-labeled analog.Because there little chemical difference between the labeled amino acidand the natural amino acid isotopes, the cells behave like the controlcell population grown in the presence of normal amino acid.

Conventional SILAC processes two culture lysates that are mixed andproteolyzed with optionally one or more stages of affinity enrichment.Unlabeled and labeled samples can be combined prior to lysis of thecells and treated as a single sample in all subsequent steps. Thisallows the experimenter to use any method of protein or even peptidepurification (after enzymatic digestion) without introducing error intothe final quantitative analysis.

SILAC methods are disclosed for example, in U.S. Pat. No. 6,391,649 toChait. In SILAC, quantitation by mass spectrometry (MS) is performed bymeasuring the relative peak intensities of the heavy-labeled and thelight-labeled isoforms of the peptides. Unlike chemical labelingtechniques (e.g. isotopically coded affinity tagging (ICAT) or iTRAQ,Applied Biosystems, Framingham Mass.), the incorporation of isotopicallyheavy amino acid is nearly 100%. The difference in the mass of theisotope in each cell pool results in two distinct, closely spaced peaksfor each protein or peptide actively produced by the samples in the massspectrum. One peak corresponds to a protein or peptide from a proteinfrom the cell pool with the normal abundance of isotopes. The other peakcorresponds to a protein or peptide from the cell pool enriched in oneor more of the isotopes. A ratio is computed between the peakintensities of at least one pair of peaks in the mass spectrum. Therelative abundance of the protein in each sample may be determined basedon the computed ratio. The protein may be identified by themass-to-charge ratios of the peaks in the mass spectrum, as well as byother means known in the art.

Up to the point of the MS, none of the steps of the Chait processdiscriminates between a protein that contains the natural abundance ofisotopes from the same protein from the enriched sample. Thus, theratios of the original amounts of proteins from the two samples aremaintained, normalizing for differences between extraction andseparation of the proteins in the samples.

With labeled cells in SILAC, one can proceed to do sub-cellularpurification of intact organelle structures or multi-protein complexes.The two samples can be combined as whole cells and a single subcellularpreparation of nuclei, mitochondria, etc., then prepares the samples(now combined) for MS. Any sample preparation bias introduced by thecomparison of two separate preparation steps (as would be the case in achemical modification method) is avoided. SILAC is thus proven as auseful tool for proteomic analysis.

An example of a typical SILAC experiment is illustrated in FIG. 1. FIG.1 depicts two identical cultures grown in parallel where one culture isgrown under media conditions that enrich the ¹⁵N isotope of an aminoacid (such as arginine) and the other is grown in conventional media.One of the cultures is challenged by an external stimulus (such asapplication of a drug). After the stimulus, the cell cultures areprocessed and lysates are produced. The lysates from each culture aremixed and proteolyzed with an enzyme such as trypsin. After somechromatography, the sample is analyzed by MS. MS analysis is capable ofresolving the sample components by mass and measuring the relativeabundance of the light and heavy isoforms of a specific peptide. Thelight and heavy isoforms will appear as two peaks differing in mass bythe difference in mass due to the heavy isotope present. Proteinsunaffected by the challenge will have a like ratio of the heavy isoformto light isoform peaks. A different ratio of the heavy isoform to lightisoform indicates which culture (challenged or control) has increased ordecreased expression of that protein.

Despite the advantages of SILAC over conventional chemical labelingtechniques for quantifying expressed proteins, there are severallimitations of the technique. As the name of the technique implies,incorporation of the heavy isotope requires protein expression in activecell cultures. Thus, the technique cannot be applied to tissue samples,biopsies or tissue slices. Another limitation is that proteinquantitation by SILAC is merely relative (i.e. the relative expressionof a specific protein in one experimental condition versus another) inlike treated parallel samples. SILAC does not provide quantitationinformation to allow comparison of concentration of different molecules,for example different proteins. Absolute concentration information isnot obtained using SILAC. Further, because SILAC does not provideabsolute quantitation, a comparison of results between experiments isdifficult to analyze.

Accordingly, MS and SILAC are useful tools. However, MS analysis doesnot lend to easy absolute quantitation, but only relative quantitationof the molecules analyzed. SILAC suffers from its ability to onlyprovide information of relative concentration comparing proteinconcentration between samples, and absolute quantity information may notbe obtained using SILAC.

SUMMARY OF THE INVENTION

The present invention provides mass spectrometry methods of quantifyinga biomolecule in a sample, which utilize isotopically-labeledbiomolecule standards. The present invention further providesisotopically-labeled biomolecule standards, such as protein standardsand methods of making the same. According to certain embodiments, thebiomolecule standards of the present invention are prepared usingrecombinant methods. The present invention further provides reagents andkits relating to the methods and products disclosed herein.

These and other features and aspects of the present invention arefurther described below. The descriptions provided are meant to describethe present invention with example embodiments. The example embodimentsare not considered as limiting the scope of the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example, with reference tothe following accompanying drawings:

FIG. 1 is a diagram illustrating a conventional SILAC procedure.

FIG. 2 illustrates a basic scheme according to certain embodiments ofthe present invention.

FIG. 3 schematically depicts certain embodiments of the presentinvention in which individual peptides that can be quantified areuniquely identified.

DETAILED DESCRIPTION OF THE INVENTION

The aspects, advantages and other features of the invention will becomeapparent in view of the following detailed description, which disclosesvarious non-limiting embodiments of the invention. The followingdescriptions are by way of illustration and not by way of limitation.

The art would be aided by tools and methods for absolute quantitation ofmass spectrometry samples, especially biomolecules subjected to MSanalysis. Furthermore, the art would be aided by tools and methodsallowing comparison of samples other than those obtained from cellculture, for example, samples from tissues, biopsies, serum, etc. Thepresent invention provides such tools and methods.

The present invention provides isotopically-labeled biomoleculestandards, which may be useful in quantification of one or morebiomolecules, such as proteins. Isotope-labeled biomolecule standards ofthe present invention may be controllably manufactured or expressed.Thus, the biomolecules are produced in culture or by other meansseparately, and then added to one or more cell lysates as a set ofinternal quantitation standards for MS analysis. Embodiments utilizingrecombinant expression result in reduced volumes of heavy isotopenecessary for each experiment; and allow a single standard productionrun to be used over time for multiple samples (thus greatly reducing thecost of the experiment).

Embodiments utilizing recombinant expression can also be used tosynthesize isotopically-labeled macromolecules (for example, nucleicacid molecules, protein molecules, polysaccharides) that can be useddirectly in MS, such as MALDI-TOF MS, to quantitate macromolecules.Large biomolecules (for example, biomolecules greater than 5 kDa,greater than 10 kDa, or greater than 20 kDa) can also be fragmented,using enzymes such as, but not limited to, proteases (including forexample serine proteases, sulfhydryl proteases, metalloproteases),phosphorylases, peptidases, diesterases, lipases, oxidoreductases,transferases, hydrolases, lyases, or isomerases. The fragments can beused to quantitate the biomolecule of interest or to determine relativeand absolute amounts of variants of macromolecules (such as variantshaving different polymer lengths, biochemical modifications, etc.).

Recombinant techniques that use engineered cells or cell extractsprogrammed with a recombinant nucleic acid template can be used tosynthesize biomolecule standards by introducing a gene that encodes abiomolecule of interest into a cell (or cell extract) or by introducinga gene that encodes an enzyme or regulator that controls the synthesisof the copy biomolecule in a cell or cell extract. These methods canresult in the synthesis of macromolecules, such as, but not limited to,proteins, nucleic acid molecules, and polysaccharides, that can beisotopically labeled during synthesis, purified, quanititated, andsubsequently fragmented to obtain a set of biomolecule standards thatcomprise copy isotopically-labeled fragments of the biomolecule ofinterest such that MS of a sample that includes a fragmented biomoleculeof interest includes multiple copy fragment standards that can be usedto quantitate biomolecule variants in the sample. For example, splicevariants of proteins or nucleic acid molecules and enzymaticallymodified biomolecules (including, but not limited to, proteolyticallyprocessed proteins, post-translationally modified proteins,poly-adenylated RNAs, etc.) can not only be detected, but quantitated byuse of a set of fragment standards generated from a macromolecule.

The present invention is advantageous over prior methods for example, inenabling accurate quantitation of biomolecules analyzed by MS by spikinga copy biomolecule standard, i.e., a biomolecule that reacts preferablybiologically and chemically like the biomolecule copied, into a sample.The copy molecule is differentiatable under MS from the biomoleculecopied, for example by the incorporation therein of an isotopic label.Thus, knowing the quantity and/or concentration of the copiedbiomolecule introduced into the MS process allows an accuratedetermination of the relative or absolute quantity of the biomoleculecopied. Alternatively, relative changes could be analyzed if the sameamount (e.g., volume) of the biomolecule copy is added to two samplesand relative peak intensities are analyzed. Comparison of concentrationsof a peptide of interest in two samples may be enabled, with or withoutdetermining an absolute concentration of the copy molecule.

The present invention can be used for quantitation of one or morepolypeptides in a sample. The methods include adding anisotopically-labeled copy polypeptide of interest of known quantity to asample that contains the polypeptide of interest (or a variant thereof,for example, a splice variant, a polypeptide generated from a differentmember of a gene family, a variant having one or more post-translationalmodifications); analyzing the sample by mass spectrometry; and comparingthe mass spectrometry peaks of pairs resulting from theisotopically-labeled copy polypeptide of interest and the polypeptide ofinterest of the sample, in which the isotopically-labeled copypolypeptide differs from the mass of the polypeptide of interest by anamount corresponding to the difference in mass between the labelingisotope and the cognate naturally-occurring isotope, taking into accountthe number of labeled isotopes incorporated into the polypeptide. Themethods further include comparing peak heights of the members of a peakpair to quantitate the polypeptide of interest in the sample, takinginto account the known mass of the added copy polypeptide.

In some embodiments, the polypeptide copy and the polypeptides of thesample are fragmented prior to MS. In these embodiments, fragment peaksare compared and used to quantitate the amount of individual fragmentsof a polypeptide of interest. In some aspects, multiple fragments of acopy polypeptide can thus be used to assess the internal consistency ofthe quantitation for a given polypeptide of interest. In other aspects,multiple fragments of a copy polypeptide can be used to determine therelative and absolute abundance of splice variants or differentiallymodified variants of a protein of the sample. In these aspects, forexample, at least one fragment generated from the copy polypeptide priorto MS is preferably within or substantially overlaps a protein domainthat is encoded by an alternatively spliced exon of a gene encoding thepolypeptide of interest, or is within or substantially overlaps a domainof the protein that can be proteolytically processed in a cell, orincludes a post-translational modification site.

In other embodiments, copy and sample polypeptides may not be fragmentedprior to MS. In these embodiments, polypeptide standards are synthesizedin engineered cells or in in vitro protein synthesis systems. Thepolypeptide standards and sample polypeptides can be analyzed, forexample, by MALDI-TOF MS to quantitate the amount of protein in asample.

The present invention is also advantageous in that it allows researchersto extend advantages of the SILAC technique (such as making use of theefficiency of heavy isotope labeling and its steadfastness with respectto changes to protein conformation, compartmentalization or aggregationstate that might alter the accessibility of reaction sites, etc.) tosamples involving non-cultured cells, for example, tissue samples,including blood, serum, semen, sputum, cerebrospinal fluid (CSF),saliva, etc, tissue slices, biopsies, aspirates, necropsies, etc.Another advantage of the present invention is the accurate quantitationof proteins that are not actively transcribed/translated, since thesample itself does not have to incorporate label for quantitation ofproteins. Cells that do not divide in culture, for example, neuralcells, can also be used for quantitation of biomolecules such asproteins. Quantitation is also possible for cells that are notcultured—for example, cells of an organism, embryos, tissues, organs, orbodily fluids. The present invention allows absolute quantitation ofproteins in addition to relative quantitation of proteins.

The present invention is also advantageous over prior methods, such asSILAC, in that it further enables use in culture as well as in tissueslices and even samples obtained from living organisms.

In describing embodiments of the present invention, specific terminologyis employed for the sake of clarity. However, the invention is notintended to be limited to this specific terminology. It is to beunderstood that each specific element includes all technical equivalentsthat operate in a similar manner to accomplish a similar purpose.Additionally, elements and features of the embodiments described can becombined with other embodiments of the invention to create newembodiments that are also included in the present invention. Further,all of the citations herein are incorporated by reference in theirentirety.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein are well known and commonly employed in the art. Where aterm is provided in the singular, the inventors also contemplate theplural of that term. Where there are discrepancies in terms anddefinitions used in references that are incorporated by reference, theterms used in this application shall have the definitions given herein.As employed throughout the disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meanings:

As used herein, “a”, “an” and “the” may mean one or more. As usedherein, “another” may mean at least a second or more.

As used herein, an “analytical result” of mass spectrometry is typicallyexpressed as a peak or collection of peaks that indicate mass of aparticle. However a peak is only one mode of indicating relation of massto charge. “Peak” is used to indicate a distinct particle separatedbased on its mass/charge ratio regardless of the mode through which thedata are expressed.

As used herein, the term “biomolecule” is intended to include anybiomolecules for which quantification may be desired, including, but notlimited to whole proteins, glycoproteins, lipoproteins, and otherpolypeptides; carbohydrates; peptides; nucleic acids, lipids, glycolyticfragments; sugar compounds; polysaccharides and the like.

A “copy” of a biomolecule may be for example, a biomolecule, such as apolypeptide that behaves in a chemical and electrostatic fashion that isessentially identical to the polypeptide copied. The copy may be longeror shorter than the copied polypeptide and/or may differ in sequence insome regions from the copied polypeptide. However, at least one fragmentof the copy, for example, a tryptic fragment, will behave ionicly andchemically as a fragment of the same region of the polypeptide copied.By way of non-limiting example, arginines synthesized with a heavynitrogen isotope will have essentially the same biological and chemicalproperties as natural arginine. When this heavy arginine is incorporatedinto a polypeptide, the arginine residue will behave the essentially thesame whether it is natural or a copy; the protein will similarly behavethe same whether containing a heavy or light isotope. Cleavage at thisresidue, for example, by a tryptic enzyme, will produce a peptidefragment of heavier mass due to the terminal high mass arginine, butwith the same chemical and ionizing characteristics as the peptidefragment from a natural peptide. For example, the tryptic enzyme may betrypsin or an enzyme with cleavage characteristics similar to trypsin,such as TRYPle™ (Invitrogen, Carlsbad, Calif.). The high masspolypeptide from which the high mass peptide is cleaved is thus for thepurposes of the present invention considered a copy of the naturalpolypeptide, although flanking regions of the copy peptide fragment mayor may not have the same characteristics as flanking regions of thenatural polypeptide. For example, as discussed further below, the copymay include a tag or marker to facilitate or monitor synthesis and/or toaid purification.

A “derivative” of a cell or cell line is a cell or cell population whoselineage can be traced to a specific cell or cell line, such as aspecific passage of the cell or cell line. An engineered cell generallywill not be considered a derivative if a chromosome altered byengineering contains a marker or lacks a marker not found or found,respectively, in the chromosome or region of the cell or cell line whosesource of derivation is questioned.

As used herein, an “engineered cell” is a cell that has an unnaturalgenome, that is, a cell whose genetic material has been altered by man.The altered genome may include an episome (such as a plasmid), or aviral construct (such as an adenoviral construct, adeno-associated viralconstruct, and alphaviral construct, a retrovial construct, etc.), thatis introduced into the cell, or a deletion of, addition to, or mutationof a genome of an organelle, or one or more chromosomes of the cell.Genetic engineering can also comprise one or more of homologousrecombination, expression or introduction of antisense RNA, expressionor introduction of RNAi, inappropriate expression of a gene, orexpression of a dominant negative form of a gene.

As used herein, an “expression product” takes on a meaning broader thanthe product of transcription and translation. An expression product canalso be a product that results from a reaction within a cell that isguided by a gene, for example, a transgene.

As used herein, the terms “heavy-isotope labeled biomolecule,” and“isotopically-labeled biomolecule” mean a biomolecule that hasincorporated in its chemical structure, an isotope of an atom that isdifferent from the predominant isotope found in nature. In context, anatom refers to a plurality of atoms having the same atomic number.

In various aspects, the invention includes mass spectrometry. As usedherein, the term “mass spectrometry” (or simply “MS”) encompasses anyspectrometric technique or process in which molecules are ionized andseparated and/or analyzed based on their respective molecular weights.Thus, as used herein, the terms “mass spectrometry” and “MS” encompassany type of ionization method, including without limitation electrosprayionization (ESI), atmospheric-pressure chemical ionization (APCI) andother forms of atmospheric pressure ionization (API), and laserirradiation. Mass spectrometers are commonly combined with separationmethods such as gas chromatography (GC) and liquid chromatography (LC).GC or LC separates the components in a mixture, and the components arethen individually introduced into the mass spectrometer; such techniquesare generally called GC/MS and LC/MS, respectively. MS/MS is ananalogous technique where the first-stage separation device is anothermass spectrometer. In LC/MS/MS, the separation methods comprise liquidchromatography and MS. Any combination (e.g., GC/MS/MS, GC/LC/MS,GC/LC/MS/MS, etc.) of methods can be used to practice the invention. Insuch combinations, “MS” can refer to any form of mass spectrometry; byway of non-limiting example, “LC/MS” encompasses LC/ESI MS andLC/MALDI-TOF MS. Thus, as used herein, the terms “mass spectrometry” and“MS” include without limitation APCI MS; ESI MS; GC MS; MALDI-TOF MS;LC/MS combinations; LC/MS/MS combinations; MS/MS combinations; etc.

“Means for synthesizing” a polypeptide, biomolecule, protein, and thelike, simply refers to tools that might be used for peptide synthesis,for example, a gene, host cell, in vitro translation system (such asInvitrogen Expressway™ Translation Systems), a buffer, a medium, aminoacids, amino acid precursors, a solid phase synthesis system, orcomponent part thereof.

As used herein, a “polypeptide” is a polymer of amino acids linked bypeptide bonds. As used herein, a “protein” is a polypeptide, that is, apolymer of amino acids. The terms “protein” and “polypeptide” are usedsomewhat interchangeably herein. While the protein may have biologicactivity, no inference of a requirement for activity is to be made. Aprotein may contain modifications to the amino acid monomers. Forexample, a protein may be glycosylated or phosphorylated. “Protein”includes protein containing molecules such as lipoproteins andglycoproteins. The terms “polypeptide” and “peptide” are used somewhatinterchangeably herein. In the art, “peptide” is simply a moleculecontaining peptide bonds. The term “peptide” is generally used todescribe fragments of a protein or polypeptide, for example, resultingfrom enzymatic or chemical digestion, for example, using a trypticenzyme or cyanogens bromide. But the term “polypeptide” may similarlyinclude fragments thereof. In analysis by mass spectrometry, apolypeptide is often split into multiple peptides, for example, two,three, four, five, six, seven or more peptide components from thepolypeptide. Analysis of any one of these peptide fragments yieldsinformation with respect to the source polypeptide. When data fromplural peptides are confirmatory, confidence in the analysis isincreased. Terms such as “polypeptide of interest”, “protein ofinterest” and “biomolecule of interest” include any source polypeptide,protein or biomolecule or peptide bond containing fragment thereof aboutwhich an investigator seeks information.

A “substantially non-radioactive isotope” is an isotope or mixture ofisotopes whose disposal is not regulated by rules designed to protectthe public from radiation exposure.

A “synthesized copy” is a copy made by any non-natural means, that is, acopy made as a result of the intervention of man. For example, synthesismay be accomplished by providing chemicals and reaction conditions insequence to produce a desired biomolecule; by providing an in vitrotranslation system, such as a cell extract in vitrotranscription/translation system; by providing a host cell (prokaryoticor eukaryotic) and a template for the host cell to use for manufacturinga biomolecule of interest such as a polypeptide; or by providingchemicals and/or catalysts, such as enzymes, to produce a desiredbiomolecule. The synthetic process may include a concentration process,a purification process, an enriching process, etc. includingphotoreactive processes to facilitate use of the synthesized copy in adesired process or analysis.

The terms “quantitation” and “quantification” and forms thereof, areused interchangeably herein.

A “transgene” as used herein, is a nonnatural gene or a gene that hasbeen translocated to a nonnatural location or environment.

Methods of Quantifying One or More Biomolecules

The present invention includes methods for quantifying one or morebiomolecules, such as proteins or other polypeptides in a sample usingmass spectrometry. Such methods may include providing at least onesample having a biomolecule of interest to be quantified; providing acopy of the biomolecule of interest, in which the copy is made using anisotope having a mass different than that of naturally-occurringisotopes of the biomolecule of interest; quantifying the biomolecule ofinterest in the copy; introducing a known quantity of the copy into saidsample; analyzing the sample by mass spectrometry; and comparingobtained mass spectrometry peak pairs resulting from the one or morebiomolecule or a fragment thereof, and the copy or a fragment thereof todetermine the quantity of the mass spectrometry peak pairs differ inmass by an amount corresponding to the isotope with the different masstaking into account the number of isotope atoms in each ion monitored.

Other embodiments of the present invention include methods forquantifying one or more biomolecules using a biomolecule standard madeusing recombinant expression. Such methods may include for example,providing at least one sample having a biomolecule of interest to bequantified; providing a copy of the biomolecule of interest, in whichthe copy is made using recombinant expression and the copy includes anisotope having a mass different than that of naturally occurringisotopes of the biomolecule of interest; quantifying the biomolecule ofinterest in the copy; introducing a known quantity of the copy into thesample; analyzing the sample by mass spectrometry; and comparingobtained mass spectrometry peak pairs resulting from the one or morebiomolecules or one or more fragments thereof, and the copy or one ormore fragments thereof to determine the quantity of the one or morebiomolecules in the one or more samples.

Samples in accordance with the present invention may include forexample, at least one component selected from the group consisting ofbiological cells, a cell supernatant, a cell extract, embryos, a celllysate, viruses, biological tissue, a tissue slice, an organ, anorganism, a collection of organisms, a portion of an organism, a biopsy,a sample of bodily fluid, a blood sample, a serum sample, and acell-free biological mimetic system.

The copy of the biomolecule of interest is labeled with at least oneisotope to form a standard. Non-limiting examples of isotopes forlabeling include ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁴S, etc. Preferably theisotope is a heavy isotope that is substantially non-radioactiveisotope. By isotope-labeled biomolecule, is meant a biomolecule that hasincorporated in its chemical structure an isotope of an atom that isdifferent from the predominant isotope found in nature. Thus, the labelis a mass-altering label. The mass of the label should be such that uponMS analysis, a peak associated with the biomolecule is distinguishablefrom a peak associated with the isotope-labeled biomolecule.

The biomolecule standard may be chemically synthesized or may takeadvantage of biological processes or molecules. The labeled atom ormolecule is incorporated into the biomolecule and spiked into a samplefor quantitation of one or more biomolecules in the sample.

According to certain embodiments, the copy of the biomolecule (e.g.,polypeptide or protein) of interest may be made by example, using freecell synthesis or an engineered cell. Engineered cells of the presentinvention may be cells derived from a cell or cell line other than acell or cell line from which the sample is obtained.

According to certain embodiments, the biomolecule standard may be madeusing recombinant methods, such as in vitro translation and synthesis inan engineered cell or a cultured cell. For example, copies of thebiomolecule may be made by expressing the biomolecule from an engineeredconstruct in a cultured cell (which can be episomal or incorporated intoa host cell chromosome), or from a cell that has been engineered(genetically modified) to overexpress or inducibly express an endogenousgene or a gene recombined into the cell's genome (recombinant cells).Generation of the reference standard for quantitative analysis ofexpressed proteins by recombinant methods has a number of advantagesover using a control cell culture. One advantage is the reduced amountand cost of isotopically-labeled amino acid. Another advantage is theability to efficiently synthesize isotopically-labeled macromolecules.Methods for making biomolecule standards using recombinant methods aredescribed further below.

The copy of the polypeptide includes at least one isotope in an aminoacid or amino acid residue of the polypeptide. According to certainembodiments, the polypeptide includes at least one substantiallynonradioactive isotope. Isotopes used in accordance with the presentinvention have a mass different than that of naturally occurringisotopes of the polypeptide of interest, and may include for example, atleast one isotope selected from the group consisting of ¹⁵N, ³C, ¹⁸O, ²Hand ³⁴S. Amino acids having mass-altering isotopes can be any aminoacids, including naturally-occurring amino acids, modified amino acids,and synthetic amino acids that can be incorporated into peptides andproteins, and are preferably naturally-occurring amino acids.Non-limiting examples of the amino acids or amino acid residues usefulfor making isotopically-labeled polypeptide copies include thoseselected from the group consisting of Arg, Lys, Asp, Glu, Met, Trp, Ser,Thr, Tyr, and Asn.

According to certain embodiments, the copy may be made using an isotopepool. For example, the labeling isotope may be present in a pool ofmolecules containing the labeling isotope as a predominant fraction ofthe pool. The isotope pool may include for example, substantiallynon-radioactive isotopes. By way of non-limiting example, the isotopepool may include at least one isotope having a mass distributiondifferent than that of naturally occurring isotopes in the polypeptideof interest. For example, the labeling isotope may be present in a poolof molecules, such as a pool of amino acids including one or more of,for example, arginine, threonine, serine, tyrosine molecules in afraction for example at least about 100%, 99%, 98%, 95%, 90%, 80%, 75%of the pool. Higher fractions are expected to provide higher qualitydata.

Certain embodiments for example, use amino acids labeled with ¹⁵N or¹³C, more preferably ¹⁵N-Arg or ¹³C-Arg. This expression will be used asan exemplary shorthand expression for the heavy-isotope labeledbiomolecules of the present invention. For example, a pool of ¹⁵N-Argwould include a significant, i.e., measurable, difference from a naturalpool of Arg. The pool of atoms, e.g. a pool where ¹⁵N predominates maybe incorporated into a biomolecule. Labeled biomolecules synthesizedusing the pool will be distinguishable, for example in MS from naturalforms of the biomolecule.

A biomolecule standard may be quantified, for example, by biochemical orspectroscopic methods as known in the art for the particular type ofbiomolecule. The biomolecule standard may then be used for example, as aquantitative internal reference for a sample cell lysate. A proteinstandard can be quantified by protein quantitation assays as they areknown in the art, for example, ELISA, Bradford assays, Lowry assays,bicinchoninic acid (BCA) assays, or modified versions of these, orfluorescence based assays, such as are commercially available (forexample, Quant iT™ Assay Kit [Invitrogen], NanoOrange® ProteinQuantitation kit [Invitrogen], EZQ™ Protein Quantitation kit[Invitrogen], FluoroProfile™ [Sigma-Aldrich]).

The biomolecule standard may then be introduced into the sample to beanalyzed. According to certain embodiments, the quantitation standard isintroduced into the sample as early in the processing of the sample aspossible. This ensures that losses or modifications induced by thefractionation process occur evenly across the sample components and thereference standard(s).

Mass spectrometry provides a rapid and sensitive technique for thecharacterization of a wide variety of molecules. In the analysis ofpeptides and proteins, mass spectrometry can provide detailedinformation regarding, for example, the molecular mass (also referred toas “molecular weight” or “MW”) of the original molecule, the molecularmasses of peptides generated by proteolytic digestion of the originalmolecule, the molecular masses of fragments generated during theionization of the original molecule, and even peptide sequenceinformation for the original molecule and fragments thereof.

A time-of-flight mass spectrometer determines the molecular mass ofchemical compounds by separating the corresponding molecular ionsaccording to their mass-to-charge ratio (the “m/z value”). Ions areaccelerated in the presence of an electrical field, and the timenecessary for each ionic species to reach a detector is determined bythe spectrometer. The “time-of-flight” values obtained from suchdeterminations are inversely proportional to the square root of the m/zvalue of the ion. Molecular masses are subsequently determined using them/z values once the nature of the charged species has been elucidated.

A particular type of MS technique, matrix-assisted laser desorptiontime-of-flight mass spectrometry (MALDI-TOF MS) (Karas et al., Int. J.Mass Spectrom. Ion Processes 78:53, 1987), has received prominence inanalysis of biological polymers for its desirable characteristics, suchas relative ease of sample preparation, predominance of singly chargedions in mass spectra, sensitivity and high speed. MALDI-TOF MS is atechnique in which a UV-light absorbing matrix and a molecule ofinterest (analyte) are mixed and co-precipitated, thus forminganalyte:matrix crystals. The crystals are irradiated by a nanosecondlaser pulse. Most of the laser energy is absorbed by the matrix, whichprevents unwanted fragmentation of the biomolecule. Nevertheless, matrixmolecules transfer their energy to analyte molecules, causing them tovaporize and ionize. The ionized molecules are accelerated in anelectric field and enter the flight tube. During their flight in thistube, different molecules are separated according to their mass tocharge (m/z) ratio and reach the detector at different times. Eachmolecule yields a distinct signal. The method is used for detection andcharacterization of biomolecules, such as proteins, peptides,oligosaccharides and oligonucleotides, with molecular masses betweenabout 400 and about 500,000 Da, or higher. MALDI-MS is a sensitivetechnique that allows the detection of low (10⁻¹⁵ to 10⁻¹⁸ mole)quantities of analyte in a sample.

Partial amino acid sequences of proteins can be determined by enzymaticor chemical proteolysis followed by MS analysis of the product peptides.These amino acid sequences can be used for in silico examination of DNAand/or protein sequence databases. Matched amino acid sequences canindicate proteins, domains and/or motifs having a known function and/ortertiary structure. For example, amino acid sequences from anuncharacterized protein might match the sequence or structure of adomain or motif that binds a ligand. As another example, the amino acidsequences identified by MS analysis can be used as antigens to generateantibodies to the protein and other related proteins from otherbiological source material (e.g., from a different tissue or organ, orfrom another species). There are many additional uses for MS,particularly MALDI-TOF MS, in the fields of genomics, proteomics anddrug discovery. For a general review of the use of MALDI-TOF MS inproteomics and genomics, see Bonk et al. (Neuroscientist 7:12, 2001).

Tryptic peptides labeled with light or heavy amino acids can be directlyanalyzed using MALDI-TOF. However, where sample complexity is apparent,on-line or off-line LC-MS/MS or two-dimensional LC-MS/MS may benecessary to separate the peptides.

MS may be capable of resolving the heavy and light isotope isoforms foreach peptide, as well as measuring the relative intensity of eachisoform.

After MS is performed, mass spectrometry peak pairs result from eachbiomolecule being analyzed and its corresponding biomolecule standardhaving a different molecular weight. The mass spectrometry peak pairsmay be compared to determine either an absolute or relative quantity ofthe one or more biomolecules in the sample. According to certainembodiments, the present invention features use of the protein standardto quantify proteins that share regions of sequence identity (such ashomologous proteins or splice variants).

Methods of the present invention may further include forming fragmentsof the biomolecule, for example, by digestion with an enzyme or cleavagewith a chemical reagent. Enzymes in accordance with the presentinvention may include for example, at least one enzyme selected from thegroup consisting of a protease (for example, a serine protease), aphosphorylase, a peptidase, a diesterase, a lipase and anoxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, and aligase.

According to certain embodiments, the at least one sample includes aplurality of samples. At least a first sample of the plurality ofsamples may have been exposed to a different set of conditions than asecond sample of the plurality of samples. Non-limiting examples ofdifferent sets of conditions may include for example, species or strainfrom which the sample is obtained, disease-state versus normal state ofan organism from which the sample is obtained; one or more of: chemicalexposure; genetic manipulation; feeding regimen; environmentaldifference; aging or developmental stage; or difference in exposure toone or more hormones, growth factors, or cytokines.

Genetic manipulation may include for example, performing geneengineering. Genetic engineering can include one or more of: homologousrecombination (for gene knockout or replacement), expression orintroduction of antisense RNA, expression or introduction of RNAi,inappropriate expression of a gene, or expression of a dominant negativeform of a gene. Chemical exposure may include for example, at least oneof exposure to a drug or metabolite thereof, exposure to a drugcandidate or metabolite thereof, addition of growth factors, andaddition of cytokines to alter the phenotype of the cell. Environmentaldifference may include for example, exposure to different temperature,UV light, O₂ pressure, etc., or the presence of at least one of a virus,a bacteria and a carcinogen.

Methods according to the present invention may include providing atleast a second sample whose polypeptide of interest is desired to bequantified; introducing a known quantity of the copy into the secondsample; analyzing by mass spectrometry the second sample to determine aquantity of polypeptides of interest or a fragment thereof in saidsecond sample; and comparing the quantity of at least one polypeptidesof interest or one or more fragments thereof of the sample to the secondsample. According to certain embodiments, the second sample may be froma preparation different from the sample.

According to certain embodiments, biomolecules of interest, such asproteins of interest, may be quantified from a cell lysate. Cells may begrown in culture or obtained from a tissue sample from an organism, forexample, the tissue sample may be a tissue slice or a biopsy, forexample a needle biopsy. The sample may be obtained for example, from ablood sample. The cell lysate is prepared from a cell population. Lysiscan be accomplished in any fashion, for example, by detergentdisruption, sonication, osmotic shock, freezing, etc. Embodiments of theinvention may include for example, quantifying proteins from a celllysate that originated from cells that expressed proteins recombinantlyor naturally in cell culture, as well as samples that originated fromplasma, serum, CSF, urine, sputum, semen, lymph, or any biologicalfluid, tissue or other sample. According to other embodiments of thepresent invention, proteins may be quantified from a cell lysate thatoriginated from cells that have been challenged by an external stimulussuch as application of a drug, radiation, or a change in theenvironment, such as a feeding schedule, temperature change, light darkcycle, etc.

According to other embodiments of the present invention, biomolecules,such as proteins may be quantified from healthy samples and compared toproteins quantified from disease samples. Comparing protein expressionpatterns between normal and disease conditions may reveal proteins whosechanges are important in the disease and thus, identify proteins whichaid diagnosis or whose modification may be of therapeutic value. Inclinical research, changes in protein expression may be usefulindicators of targets of treatments, efficacy of treatments ormonitoring unknown side effects of treatments. In basic research,changes in protein expression may be useful indicators of responses of acell to experimental manipulation. Methods of detecting proteinexpression profiles may also have important applications in, for exampleand without limitation, tissue typing, drug screening, forensicidentification, and clinical diagnosis.

Certain embodiments of the invention are directed to drug or toxicologyscreening. For example, a sample from a culture, a tissue or an organismexposed to a drug, a drug metabolite, a drug candidate or a drugcandidate metabolite, may be analyzed to test efficacy, toxic effect,alternate uses, etc.

Another aspect of the present invention features quantifying theabundance of a biomolecule that is a non-protein biological metabolicproduct (i.e. such as lipids, fatty acids, DNA, steroids, carbohydrates,and the like). In this example, a heavy-isotope labeled biologicalmolecule may be synthesized and then spiked into a lysate for thepurpose of measuring the relative abundance to the naturally-occurringlight-isotope isoforms. The abundance of the naturally occurring lightform can then be determined.

Another aspect of the present invention may allow monitoring effects ofstimuli or conditions, such as feed regimens, growth factors, cytokines,temperatures, pH, etc., for effects such as phenotypic effects on acell, organism, or tissue. For example, differentiation ordedifferentiation, for example of adult or stem cells, e.g.,differentiated cells or stem cells, hematopoietic stem cells, neuralstem cells, cardiac stem cells, etc, may be monitored fordifferentiation or dedifferentiation markers. Cells in this aspect aswell as other aspects may be human or other animal, for example, mammal,insect or fish. Rodent cells such as rat or mouse may be used. Foodproducing organisms such as plant or meat producing animals are likewisesuitable sources for sample material and/or cells. Corn, grain, wheat,rice, ovine, porcine, equine, etc. tissues may supply samples. Samplesmay be used for possible commercial development or for experimentalconvenience.

Certain embodiments of the present invention include using at least onemanufactured protein, for example, a recombinantly expressed andheavy-isotope labeled reference protein and/or set of proteins. A singleprotein may be investigated. However, multiple proteins or polypeptidescan be manufactured to contain a heavy isotope. Any or all of themanufactured proteins can be spiked into a sample. In standard practicethe peptides will each be resolved during MS analysis so that if themass/charge profile of peptides from a protein or polypeptide is known,the quantity of the protein spiked into the sample can be used tocalculate the quantity of the corresponding peak from the sample, afterdilutions from introduction of the reference standard are accounted for.

Yet another aspect of the present invention features quantifying theabundance of biological derivatives, for example, biomolecules acted onby an environmental substance, for example a toxic substance. A knownderivative may be synthesized, and used to spike one or more samples. Aderivative may incorporate component atoms of the environmentalsubstance or may result from interaction of a cell with theenvironmental substance.

Another aspect of the present invention features aiding identificationof “ion-pairs” using “High/Low” mass spectrometry analysis. In“High/Low” MS, the user programs the MS instrument to alternate theexperimental conditions during electrospray ionization such that thevoltage cycles between high voltage settings (which induce fragmentationrandomly along the backbone of a peptide) and low voltage settings(which preserve the integrity of the peptide backbone). Thus, during thelow voltage settings, the instrument is analyzing the parent ions, andin during high voltage conditions these parent ions are all beingfragmented simultaneously. This MS technique allows a user to analyzepeptides as they elute from a liquid chromatography separation with avery high duty cycle. Because the “High/Low” technique does not usetraditional MS/MS methods where one stage of MS selects a precursormass, and a second stage of MS analyzes fragment ions generated by acollision cell, computational algorithms are required to decipher thedata being generated. The present invention could aid the identificationof “ion-pairs” that are separated by a set mass difference imparted bythe number of heavy isotopes. These “ion-pairs” can assist indifferentiating low-abundance signals from the background.

Yet another aspect of the invention features a quantitation method forassaying the quantity of a sample prepared, for example for SILAC, usinga heavy isotope. This aspect features a non-heavy isotope that will actas a copy of the heavy isotope-labeled SILAC sample. In this aspect, thestandard will appear as a lower mass peak compared to the high massisotope sample.

Methods of Making Isotopically-Labeled Biomolecule Standards

The present invention further includes methods of makingisotopically-labeled biomolecule standards using molecular biotechnologytechniques, such as recombinant expression. Such methods may include forexample, methods of making isotopically-labeled biomolecule standards,such as protein standards.

According to certain embodiments, a copy of the biomolecule of interestmay be made by synthesizing the biomolecule in engineered cells. In someembodiments, the engineered cells have been genetically manipulated tooverexpress or inappropriately express one or more enzymes that the celluses to synthesize the biomolecule and/or one or more regulators of thesynthesis of the biomolecule. A gene that encodes a biosynthetic enzymeor regulator can be introduced into a host cell, for example, optionallyunder the control of a regulatable promoter. The cell, which can beprokaryotic or eukaryotic, can be cultured in media that containsisotope labels (for example, heavy isotope labels) that are present inprecursor molecules such as, but not limited to, acetyl CoA, sugars(such as hexose and pentose sugars), cholesterol, amino acids,nucleobases, etc. The cultured host cells can then be induced tosynthesize the isotope-labeled biomolecule of interest.

In other embodiments, the engineered cells can be engineered to includea gene encoding the biomolecule of interest, such as a nucleic acidmolecule or protein. The introduced gene may include gene regulatorysequences directing expression of the gene. The cell, which can beprokaryotic or eukaryotic, can be cultured in media that containsisotope labels (for example, heavy isotope labels) that are present inprecursor molecules such as, but not limited to, sugars (such as hexoseand pentose sugars), amino acids, nucleobases, etc. The cultured hostcells can then be induced to synthesize the isotope-labeled biomoleculeof interest.

In some embodiments, the biomolecule of interest is a protein(polypeptide), and host cells are engineered to contain a nucleic acidconstruct encoding the polypeptide of interest. The host cells can beprokaryotic or eukaryotic. The cells can be bacterial cells, forexample, E. coli cells, Bacillus spp. cells (e.g., B. subtilis and B.megaterium cells), Streptomyces spp. cells, Erwinia spp. cells,Klebsiella spp. cells, Serratia spp. cells (particularly S. marcessanscells), Pseudomonas spp. cells (particularly P. aeruginosa cells), andSalmonella spp. cells (particularly S. typhimurium and S. typhi cells);yeast cells (e.g., Saccharomyces cerevisiae cells and Pichia pastoriscells); insect cells (e.g., Drosophila (e.g., Drosophila melanogaster),Spodoptera (e.g., Spodoptera frugiperda Sf9 and Sf21 cells) andTrichoplusa (e.g., High-Five cells); nematode cells (e.g., C. eleganscells); avian cells (for example, UMNSAH-DF1 cells, QT6 cells, QT-35cells); amphibian cells (e.g., Xenopus laevis cells); reptilian cells;and mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK,Per-C6, Bowes melanoma and HeLa cells). According to certainembodiments, cells may be selected from yeast cells, mammalian cells,marsupial cells, avian cells, insect cells, fish cells, amphibian cells,reptile cells, and nematode cells. Photosynthetic bacteria can also beused for protein production, and include, but are not limited to, greennon-sulfur bacteria (e.g., Choroflexus spp. (e.g., C. aurantiacus),Chloronema spp. (e.g, C. gigateum)), green sulfur bacteria (e.g.,Chlorobium spp. (e.g., C. limicola), Pelodictyon spp. (e.g., P.luteolum), purple sulfur bacteria (e.g., Chromatium spp. (e.g., C.okenii)), and purple non-sulfur bacteria (e.g., Rhodospirillum spp.(e.g., R. rubrum), Rhodobacter spp. (e.g., R. sphaeroides, R.capsulatus), Rhodomicrobium spp. (e.g., R. vanellii)).

The recombinant biomolecule copy, such as a protein copy may, forexample, usefully be prepared by expression of the biomolecule in hostcells. After insertion of the nucleic acid molecule or vector into ahost cell (i.e., transformation of the host cell), the recombinantproteins may then be produced by expression in the host cells, andsubsequently purified. In many cases, it may be preferred to synthesizea polypeptide quantitation standard in a cell type that canpost-translationally modify the polypeptide of interest in the samemanner as the source of the sample. In some embodiments, in which thesource of a sample to be analyzed by MS is mammalian cells or tissue,the host cells used for synthesizing the biomolecule copy are eukaryoticcells, and in some preferred embodiments the host cells are mammalian,avian, or insect cells.

In other embodiments, the proteins may be expressed as inclusion bodiesin bacterial host cells as described, for example, in PCT InternationalPublication No. WO98/30684. Following rupture of the cells, inclusionbodies may be separated from cellular debris using suitable separationtechniques such as centrifugation. Proteins contained in the inclusionbodies are subsequently solubilized using a denaturing agent and may be,if attached as a fusion protein to an inclusion partner protein, treatedwith a cleavage agent to remove the partner protein. In some cases, thedenatured protein may be renatured prior to further purification.

In some embodiments, the recombinant proteins may be expressed incell-free system. Such systems may facilitate, for example, theincorporation of the isotope labels into the expressed proteins. Incertain embodiments, proteins expressed in a cell-free system arelabeled using heavy isotopes.

Bacterial systems provide convenience and high-yields but not allexpressed proteins may be stably expressed in bacterial systems. Inaddition, some extracts of eukaryotic cells may have post-translationalmodification systems that can be desirable for producing copypolypeptides. Thus, an in-vitro eukaryotic or prokaryotic expressionsystem may be used for some proteins. A gene fragment may be expressedin a system where the buffer and/or media is depleted of one or morespecific amino acids so that a low abundant isotope such as a¹⁵N-enriched amino acid may be introduced for incorporation into theexpressed protein.

In some embodiments, the recombinant proteins of the instant inventionmay be expressed as part of a chimeric or multimeric protein. Arecombinant chimeric protein comprises protein sequences derived fromdifferent source proteins. A recombinant multimeric protein can includemultiple repeats of one or more protein sequences, or can comprise thesequence of an entire protein multimerized in tandem.

The present invention includes selecting the gene or encoding thesequence of the protein of interest. This sequence may be obtained, forexample, from one of many sequence databases (i.e. SwissProt, NCBi,etc.). Genes may also be obtained from libraries or commercial banks.Primers may be designed that target to a specific region of the sequencethat is unique so that only a single gene fragment is amplified. Theprimers can optionally include promoter sequences (for example, T7, T3,or SP6 promoter sequences for in vitro translation), restriction sites,recombination sites, or sequences encoding peptide tags. These primersmay then be used to amplify a gene fragment from a cDNA library or othersource (such as Open Reading Frame (ORF) library, if differentiationbetween splice variants is a requirement of the analysis).

The amplified gene may, for example, then be subcloned into anexpression vector where the gene is transcribed and translatedrecombinantly. Subcloning may be performed by any number of possiblemethods available in the art. The polymerase chosen for thisamplification reaction will depend on the subcloning scheme. Severalsubcloning kits for PCR products are available commercially.

This transcription/translation or other synthesis may be performed inculture or using a cell-free in-vitro method where the buffers and/ormedia are depleted of the naturally occurring light-isotope amino acid,and enriched with a heavy-isotope labeled substrate, for example, alabeled amino acid, sugar, lipid, etc. whose metabolism is desired to beinvestigated.

The fused vector may be used to transform a cell, for example, acompetent bacterial cell, a eukaryotic cell or introduced into a cellfree in vitro translation preparation. Bacterial cells will notglycosylate, so it may be preferred to transform a cell, for example, aeukaryotic cell, capable of glycosylation or a eukaryotic cellpreparation capable of glycosylation. Preferably, the glycosylationpattern will be similar to or identical to a glycosylation pattern ofthe protein in the cell type under investigation.

Cell extracts for in vitro protein synthesis systems can be prokaryoticor eukaryotic. The cells used for making an extract can be bacterialcells, for example, E. coli cells, Bacillus spp. cells (e.g., B.subtilis and B. megaterium cells), Streptomyces spp. cells, Erwinia spp.cells, Klebsiella spp. cells, Serratia spp. cells (particularly S.marcessans cells), Pseudomonas spp. cells (particularly P. aeruginosacells), and Salmonella spp. cells (particularly S. typhimurium and S.typhi cells); yeast cells (e.g., Saccharomyces cerevisiae cells andPichia pastoris cells); insect cells (e.g., Drosophila (e.g., Drosophilamelanogaster), Spodoptera (e.g., Spodoptera frugiperda Sf9 and Sf21cells) and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C.elegans cells); avian cells (for example, UMNSAH-DF1 cells, QT6 cells,QT-35 cells); amphibian cells (e.g., Xenopus laevis cells); reptiliancells; and mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127,BHK, Per-C6, Bowes melanoma and HeLa cells). Photosynthetic bacteria canalso be used for protein production, and include, but are not limitedto, green non-sulfur bacteria (e.g., Choroflexus spp. (e.g., C.aurantiacus), Chloronema spp. (e.g., C. gigateum)), green sulfurbacteria (e.g., Chlorobium spp. (e.g., C. limicola), Pelodictyon spp.(e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium spp.(e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhodospirillumspp. (e.g., R. rubrum), Rhodobacter spp. (e.g., R. sphaeroides, R.capsulatus), Rhodomicrobium spp. (e.g., R. vanellii)). Plant cellextracts, such as wheat germ extracts, can also be used.

In vitro protein synthesis systems are known in the art and commerciallyavailable (for example the Invitrogen Expressway™ in vitro translationsystems). The in vitro protein synthesis systems can be programmed byRNA or DNA (in vitro transcription/translation systems). Synthesis ofRNA from a DNA template is well known in the art and commercial kits areavailable. Such in vitro translation systems can include one or moreisotopically-labeled amino acids for incorporation into proteins.

Vectors expressing the proteins used in the compositions of the presentinvention may be, for example, phage, plasmid, or phagemid vectors.Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids. The DNA insertencoding a protein may be operatively linked to an appropriate promoterknown to the skilled artisan.

Expression vectors of the present invention may include at least oneselectable marker. Such markers may include for example, tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.

The vector may have been designed such that a fusion protein includingthe protein of interest and an affinity tag will be generated upontranslation. For bacterial expression, selection for bacteria thatcontain the vector may be achieved by growth in antibiotic media.

After the desired gene fragment is subcloned, the fragment may beexpressed, for example in a host expression cell, e.g., in a bacterialexpression system, a eukaryotic expression such as a yeast cell, or ahigher cell. When a host cell is used, it may be for example, a cell ofthe same tissue, species, genus, order, family or class as the source ofthe sample. Posttranscriptional modifications such as alternativesplicing or posttranslational modifications such as glycosylations,phosphorylations, etc. may thus be maintained between samples andstandards. According to certain embodiments, the cell is derived from acell or cell line different from that of the sample. Representativeexamples of host cells appropriate for the expression of the instantrecombinant proteins include, but are not limited to, bacterial cellssuch as E. coli, Streptomyces spp., Erwinia spp., Klebsiella spp.,Salmonella typhimurium, and Caulobacter crescentus. Preferred as a hostcell is E. coli, and particularly preferred are E. coli strainsBL21(DE3), BL21-Star™ (DE3), BL2′-Al™, TOP10, LMG194, GI724, which areavailable commercially (Invitrogen Corp., Carlsbad, Calif.). Other E.coli strains that may be used may include DH 10B cl and STBL2. In somecases the strains further contain other plasmids, such as, for example,pLysS or pLysE, for reduction of basal expression of recombinantproteins or for other reasons. Other examples of appropriate host cellsfor use in the expression of the recombinant proteins of the inventioninclude yeast cells, insect cells, and mammalian cells.

The expressed fusion protein may then be isolated, by any suitablemethod. Expressed proteins may be purified by any of a variety ofprotein purification techniques that are well known to one of ordinaryskill in the art. For example, the fusion protein may be isolated andpurified using metal-chelate chromatography, antibody affinitychromatography or other techniques known to those skilled in the art.Suitable techniques for purification may include, but are not limited toat least one of the following techniques: ammonium sulfate or ethanolprecipitation, acetone precipitation, acid extraction, electrophoresis,isoelectric focusing (“IEF”), immunoadsorption, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, immunoaffinity chromatography,size exclusion chromatography (“SEC”), liquid chromatography (“LC”),high performance LC (“HPLC”), fast protein LC (“FPLC”), hydroxylapatitechromatography, lectin chromatography, immobilized metal affinitychromatography (“IMAC”), metal chelation chromatography, and continuousflow electrophoresis (“CFE”).

Inclusion of an affinity tag, such as one or more histidine tags, mayallow quick and easy purification of the expressed protein by affinitycapture, for example with a metal chelate resin. A tag is not necessaryhowever. Any desired tag including an immune recognized tag may be used.Non-limiting examples of affinity tags that may be used in accordancewith the present invention include for example, V5, FLAG purificationtag, c-myc or a poly-His tag (e.g., HH, HHH, HHHHHH or any polymer orcombination thereof), polyHistidine, hemeagglutinin, GST, biotin, GFP,and polycysteine (for example, tetracysteine), etc. The tag may alterthe mass of only one peptide, for example a C-terminal peptide. Otherpeptides will be essentially identical in sequence and chemistry to thenative protein, but differ in mass due to the use of the non-naturallypresent isotope.

The copy protein or polypeptide may be fragmented to produce multiplefragments for MS. For example at least two, three, four, preferably atleast four, five, six or seven and more preferably at least eight nine,ten or more peptide fragments may result. Peptide fragments can beformed by any fragmentation process, for example by biological (such asenzymatic, e.g., tryptic) processes or by chemical processes (e.g.,chemical cleavage. Tryptic enzymes and cyanogen bromide are preferredfragmentation instruments. Although a quantity can be assayed using asingle peptide fragment confidence increases as more data is available.For example, additional fragments increase confidence that the copy isbehaving as the copied molecule in fragmentation and MS and allow foraveraging to increase confidence in the absolute values obtained.

Once purified, the biomolecule standard may be quantified by biochemicalor spectroscopic methods. The concentration of the biomolecule can beassayed by one of many different commercially available methods, e.g., aLowry assay, or any method chosen by the user.

The resulting biomolecule is an isotopically-labeled biomoleculestandard. The standard may then be used for example, as a quantitativeinternal reference for a sample cell lysate.

Isotopically-Labeled Biomolecule Standards

The present invention is also directed to isotopically-labeledbiomolecule standards, such as protein standards produced usingmolecular biology techniques. Such techniques may include techniquesdisclosed herein, such as recombinant techniques, or other techniquesknown by those skilled in the art.

In some embodiments, an isotopically-labeled MS quantitation standard ismade by expressing a gene encoding a polypeptide of interest in acell-free protein synthesis system, in which one or moreisotopically-labeled amino acids is present in the cell-free synthesissystem. In some embodiments, an isotopically-labeled MS quantitationstandard is made by expressing a gene encoding a polypeptide of interestin cultured cells, in which one or more isotopically-labeled amino acidsis present in the cell culture media. In preferred embodiments, the copypolypeptide may be fragmented, such as by proteolysis with a protease orchemical reagent, prior to MS analysis, using the same protease orchemical reagent used to fragment polypeptides of the sample.

In Vitro Translation System

The present invention further provides in vitro translation systems,which may include a population of biomolecule standard precursormolecules comprising at least one atomic isotope that under massspectrometry produces at least one peak distinguishing said biomoleculestandards from otherwise identical biomolecules lacking said at leastone atomic isotope; and systemic components sufficient for translationcomprising a catalyst and biomolecule precursor molecules to effecttranslation of said biomolecule.

Kits

The present invention includes kits for performing the methods, kits forproducing the products of the present invention, and/or kits thatinclude one or more products of the present invention. Accordingly, kitsof the present invention may include any components that may be ofassistance in performing such methods or producing such products. By wayof non-limiting example, kits in accordance with the present inventionmay include kits for quantifying a biomolecule by mass spectrometry orkits for producing isotopically-labeled biomolecule or proteinstandards.

Kits in accordance with the present invention may include means for orone or more components for synthesizing isotopically-labeled biomolecule(e.g., protein) standards, for example using recombinant expression. Byway of non-limiting example, such kits may include one or morecomponents for performing in vitro translation, such as an in vitropeptide synthesis system. In vitro peptide synthesis systems may includefor example, an extract selected from the group consisting of abacterial extract, a eukaryotic extract, a plant extract, a mammalianextract, and an insect cell or arthropod extract, and one or moreisotopically-labeled amino acids.

In another example, kits can include media, and isotopically-labeledamino acids for production of standards in cell culture. Kits may alsoinclude host cells. Kits may further include a template instruction forsynthesizing a biomolecule standard.

Kits according to on aspect of the present invention include at leastone amino acid containing one or more atomic isotopes that are differentfrom naturally occurring isotopes. Non-limiting examples of amino acidsin accordance with the present invention include one or more amino acidsselected from the group consisting of Arg, Lys, Asp, Glu, Met, Trp, Ser,Thr, Tyr, and Asn. Non-limiting examples of isotopes in accordance withthe present invention include one or more isotopes selected from thegroup consisting of ¹⁵N, ¹³C, ¹⁸O, ²H and ³⁴S.

In addition to an isotopically-labeled amino acid, in one aspect kits ofthe present invention can include one or more of: a culture medium forin vitro culture; a template instruction for synthesizing a biomoleculestandard; a cell lysis reagent; a resin for purifying a biomoleculestandard; a protein quantitation reagent; an enzyme or chemical reagentcapable of cleaving said biomolecule standard; a host cell; a vector;and a polymerase.

Media provided in a kit for growing cells can include one or moreisotopically-labeled amino acids, or the one or moreisotopically-labeled amino acids can be provided separately. Media forcell culture for producing MS quantitation standards can be depleted inone or more amino acids, such as one or more amino acids that isprovided in the kit in isotopically-labeled form. The kit can alsoinclude a cell lysis reagent, such as a buffer or detergent solutionand/or one or more enzymes.

In another aspect of kits of the invention, in addition to anisotopically-labeled amino acid, kits can include one or more of: an invitro protein synthesis system; a template instruction for synthesizinga biomolecule standard; a cell lysis reagent; a resin for purifying abiomolecule standard; a protein quantitation reagent; an enzyme orchemical reagent capable of cleaving said biomolecule standard; a hostcell; a vector; and a polymerase.

The in vitro protein synthesis system comprises a cell lysate, which canbe a prokaryotic or eukaryotic cell lysate, as described herein. Theextract can be, for example, a bacterial extract, a eukaryotic extract,a plant cell extract, a mammalian cell extract, or an insect cellextract. The kit can further include at least one buffer for in vitroprotein synthesis. A buffer for translation can include one or moresalts, buffering compounds, nucleotides, amino acids, enzymes, or energysources. Preferably an in vitro synthesis buffer that includes aminoacids is depleted in one or more naturally-occurring amino acids that isprovided in isotopically-labeled form.

Kits for production of MS protein quantitation standards using in vitroprotein synthesis or cell culture systems can further include a resinfor purifying a biomolecule standard, such as a protein standard. Theresin can be, for example, chromatography media for affinitypurification, and can included, without limitation, an Ni-NTA agarosematrix, or a matrix that includes bound maltose, calmodulin, biotin, anantibody, protein A, or other affinity capture reagents. A proteinquantitation reagent included in a kit can include, without limitation,a BCA reagent, a Lowry assay reagent, a Bradford assay reagent, an ELISAreagent, or a reagent for fluorometric detection and quantitation.

Kits in accordance with the present invention may include an enzyme orchemical preparation capable of cleaving a biomolecule standard. Enzymesin accordance with the present invention may be for example, trypsin,Endo-Lys-C, Endo-Glu-C, AspN protease, yeast peptidase, V-8 protease,pepsin, subtilisin, proteinase lc, and tobacco etch virus protease, orTRYPle.™ Chemical preparation in accordance with the present inventionmay include for example, Cyanogen Bromide.

The kits can further include host cells for the production ofpolypeptide standards, as provided herein. The kits can further includea vector. A vector can be an expression vector that includes expressionsequences for transcription and/or translation. The vector can include atag sequence, such as but not limited to a sequence encoding apolyHistidine tag, a FLAG tag, a c-myc tag, a hemeagglutinin tag, GST,biotin, GFP, polycysteine, and tetracysteine. The kit can furtherinclude a polymerase, such as but not limited to a high temperaturepolymerase for nucleic acid amplification reactions, such as Taq, Pfu,or Pfx polymerase.

In another aspect, kits in accordance with the present invention mayinclude a population of biomolecule standard precursor molecules, whichmay include at least one atomic isotope that under mass spectrometryproduces at least one peak distinguishing the biomolecule standards fromotherwise identical biomolecules lacking said at least one atomicisotope. Such kits may further include for example, a catalyst.

According to another aspect of the invention, kits may includeisotopically-labeled biomolecule standards. The biomolecule standardsmay include at least one atomic isotope that under mass spectrometryproduces at least one peak distinguishing the biomolecule standard fromotherwise identical biomolecules lacking the at least one atomicisotope.

Kits in accordance with the present invention may also include a devicefor quantifiable spiking or introducing a biomolecule standard to alysate.

The kits can further optionally include a MALDI matrix, such as, forexample sinnapinic acid or alpha-cyano-4-hydroxycinnamic acid (CHCA).

In some embodiments, kits of the invention may further comprise massspectrometric probes.

Reagents

The invention further provides reagents useful for performing any of themethods described herein. In certain aspects, a reagent according to theinvention includes an isotopically-labeled biomolecule standard. Thestandard may be a subsequence of a known biomolecule and can be used toidentify the presence of and/or quantify the biomolecule in a sample,such as a cell lysate. In other aspects, a pair or more of reagents areprovided, which may include at least two isotopically-labeledbiomolecule standards. One or more of the biomolecule standards inreagents of the present invention may optionally be modified.

Engineered Cells

The present invention further includes an engineered cell, whichincludes a transgene; and an expression product of the transgene, theexpression product comprising at least one substantially non-radioactiveisotope that under mass spectrometry produces at least one peakdistinguishing the expression product from natural components of thecell.

The following examples illustrate non-limiting embodiments of theinvention. The examples set forth herein are meant to be illustrativeand should not in any way serve to limit the scope of the claimedinvention. As would be apparent to skilled artisans, various changes andmodifications are possible and are contemplated within the scope of theinvention described, and may be made by persons skilled in the artwithout departure from the spirit of the invention.

Example 1

An example of a method of quantitation of a biomolecule in accordancewith the present invention is shown in FIG. 2. According to theseembodiments, the method includes selecting a biomolecule of interest,for example, a protein, or protein family, a lipid, a carbohydrate, orcombination thereof. In FIG. 2, the biomolecule is a protein.

After the protein of interest is selected, a copy of the protein is madeinto a standard. Panel A describes the method for isolation of genes ofinterest and subcloning. According to the embodiments depicted in FIG.2, the user selects a gene encoding the sequence of the protein ofinterest. Primers may be designed that target to a specific region ofthe sequence that is unique so that only a single gene fragment isamplified. These primers may then be used to amplify a gene fragmentfrom a cDNA library.

The polymerase chosen for this amplification reaction will depend on thesubcloning scheme. By way of non-limiting example, a polymerase that hasterminase activity may be desired so that poly-adenosine tails aregenerated at the 3′ end of the amplified gene fragment. Thesepoly-adenosine tails are complimentary to the ends of a linearizedexpression vector with poly-thymidine 5′ tails. A topoisomerase enzymefuses the gene fragment to a vector. Alternatively, the primer designmay include a complimentary sequence to an expression vector (as shownin FIG. 2, panel A).

After subcloning by any number of possible methods available in the art(several subcloning kits for PCR products are available commercially),the fused vector may then be used to transform a cell, for example, acompetent bacterial cell, a eukaryotic cell or introduced into a cellfree in vitro translation preparation. In vitro systems by their natureproduce fewer biomolecules than engineered cell systems as the in vitrosystems do not synthesize all biomolecules that a living cell needs formaintenance. Bacterial cells will not glycosylate, so it may bepreferred to transform a cell, for example, a eukaryotic cell, capableof glycosylation or a eukaryotic cell preparation capable ofglycosylation. Preferably, in these embodiments the glycosylationpattern will be similar to or identical to a glycosylation pattern ofthe protein in the cell type under investigation. Also preferably thevector has been designed such that a fusion protein including theprotein of interest and an affinity tag will be generated upontranslation. For bacterial expression, selection for bacteria thatcontain the vector may be achieved by growth in antibiotic media (thevector confers resistance via a gene that is co-subcloned as illustratedby panel A of FIG. 2).

After the desired gene fragment is subcloned, the fragment may beexpressed, for example in a host expression cell, e.g., in a bacterialexpression system, a eukaryotic expression such as a yeast cell, or ahigher cell; or in an in-vitro translation system.

Bacterial systems provide convenience and high-yields but not allexpressed proteins may be stably expressed in bacterial systems. Thus,an in-vitro eukaryotic or prokaryotic expression system may be used forsome proteins. A gene fragment may be expressed in a system where thebuffer and/or media is depleted of one or more specific amino acids sothat a low abundant isotope such as a ¹⁵N-enriched amino acid may beintroduced for incorporation into the expressed protein. Inclusion of anaffinity tag may allow quick and easy purification of the expressedprotein by affinity capture, for example with a metal chelate resin.

Because the protein expressed will normally be fragmented, for exampleby enzymatic (e.g., tryptic) or chemical digestion, many peptides willresult. The tag preferably will alter the mass of only one peptide, forexample a C-terminal peptide. Other peptides will be essentiallyidentical in sequence and chemistry to the native protein orpolypeptide, but differ in mass due to the use of the non-naturallypresent isotope. According to the example depicted in FIG. 2, theproduct is purified using Ni²⁺-NTA agarose. Upon purification, theconcentration of protein can be assayed by one of many differentcommercially available methods, e.g., a Lowry assay, or any methodchosen by the user. An embodiment of this process is shown in FIG. 2,panel B.

FIG. 2, part C illustrates an embodiment of the present invention wherea plurality of pools of cells are analyzed in parallel (these pools maybe comprised of cell cultures or a plurality of tissue samples). Atleast one set of cells may be exposed to an external stimulus ormanipulation. Subsequently, lysates of the cell pools are generated. Aknown concentration of the protein standard isolated for example,according to the embodiments shown in panels A and B of FIG. 2, isspiked into each cell lysate.

Following proteolysis, the lysate components are processed for MS. Forexample, they may be separated by chromatography and fed to MS. In thisexample, the concentration of the light-isotope isoform is determined bycomparing the relative intensity to its heavy-isotope isoform. Becausethe absolute concentration of the heavy-isotope isoform is known, theabsolute concentration of the light-isotope isoform may be determined.One assumes that because the heavy and light isotope isoforms arechemically essentially identical, and because the heavy labeled isoformwas spiked into the sample prior to any fractionation, that recovery ofboth isoforms from the fractionation is essentially identical.

In this example, to compare the expression of proteins in plurality ofpools, for example, a control cell pool and an experimentally challengedcell pool, two MS analyses would be performed. Using the presentinvention, however, each run will not require a control sample becausethe absolute concentration can be determined. Multiple MS analyses arewithin the capability of normal operations, for example, operations of aMS core lab.

Example 2

According to this example, a protein standard is made as in Example 1.In this example, a known amount of the protein standard, although notquantified is added to at least two of the cell pools. Relativeconcentrations between pools with known amounts of standards may thus beobtained. While this example does not provide absolute concentrationsfor biomolecules, this example still has the advantages that parallelprocessing of samples is not required as with conventional SILAC and anysample source, not just cultured cells can be used to obtain data.

Following proteolysis, the lysate components are processed for MS. Forexample, they may be separated by chromatography and fed to MS. In thisexample, the concentration of the light-isotope isoform is determined bycomparing the relative intensity to its heavy-isotope isoform. Becausethe amount of the heavy-isotope isoform is known in each of the pools,the relative concentrations of the light-isotope isoform may bedetermined. In these embodiments, to compare the expression proteins ina first cell pool and a second cell pool, two MS analyses must beperformed versus one by conventional SILAC. However, each run will notrequire a control sample because the amount of added standard is known.Multiple MS analyses are within the capability of normal operations, forexample, operations of a MS core lab.

Example 3

Further aspects of the present invention feature analyzing the relativeabundance of multiple biomolecules, for example, proteins,simultaneously. In this example, there are for example several methodsof generating multiple recombinantly expressed heavy-isotope labeledreference proteins.

For example, the practitioner may design multiple primers to multipleunrelated genes and amplify, subclone, and express these simultaneously.The final purified protein sample will contain a pool of multipleproteins in an unknown ratio. A quantitative amount of this pool may bespiked into the challenged cell lysates and the control cell lysate, andthe relative abundance of proteins may be determined as discussedpreviously with respect to panel C of FIG. 2. In this example, only therelative abundance of each protein may be determined. However, theresults of multiple experiments may be compared if a quantitative amountof the same reference protein pool was used.

In a second method, a practitioner may simply amplify, subclone, andexpress multiple genes in separate reactions. After quantifying theresulting proteins, the user may pool these in a known ratio. The usermay then spike this pool into the challenged cell lysates and thecontrol cell lysate, and the relative abundance of proteins may bedetermined as discussed previously with respect to panel C of FIG. 2. Inthis iteration, both the relative and absolute abundance of each proteinmay be determined.

In yet another method, the practitioner may design a single set ofprimers that target a DNA sequence region that is essentially identicalamongst a family of genes. From a single PCR reaction of a cDNA library,a number of genes, gene fragments, isotypes, splice and/or sequencevariants may be amplified. These products may then be subcloned, andexpressed substantially simultaneously. The final purified proteinsample will contain a pool of multiple proteins in an unknown ratio. Aquantitative amount of this pool may be spiked into the challenged celllysates and the control cell lysate, and the relative abundance ofproteins may be determined as discussed previously with respect to panelC of FIG. 2. In this example, only the relative abundance of eachprotein may be determined. However, the results of multiple experimentsmay be compared if a quantitative amount of the same reference proteinpool was used.

A variation of the above methods includes quantitation of one or moreproteins or polypeptides in the pool. In this variation, both therelative and absolute abundance of each of the quantified proteins andpolypeptides may be determined. Quantitation of each component of thereference standard pool may be determined. This may be accomplished bypurification of each of the components followed by a quantitative assayusing any of the common techniques known to those in the art or by anyquantitation method available for the individual or class of protein orpolypeptide.

Example 4

Another aspect of the present invention features quantifyingbiomolecules using unique multiple heavy-isotope labeled referenceproteins. Multiple biomolecules can be differentially labeled so thatfragments of different biomolecules can be individually identified. FIG.3 illustrates this aspect of the invention. According to FIG. 3, a setof biomolecules, such as a set of proteins may be separately generated.For example, a set of genes may be selected and engineered into separatecells for differential expression. Each biomolecule may be labeled withan amino acid that contains a unique number of heavy-isotopes; forexample, two, three, four or more proteins can be synthesized withdifferent mass differences (e.g., one with heavy nitrogen, a second withhydrogen and carbon; a third with nitrogen, carbon, a heavy oxygen,etc.); thus a unique molecular mass, and unique mass shift from thenaturally occurring light-isotope isoform.

FIG. 3A exemplifies three conditions with a unique isotope mix, anarginine with two ¹⁵N atoms, an arginine with four ¹⁵N atoms, and anarginine with four ¹⁵N and two ¹³C atoms. As would be apparent to thoseskilled in the art different residues might be used and differentcombinations of isotopes are possible in accordance with the presentinvention.

FIG. 3 shows three proteins, A, B and C with fragments that differ fromnative fragments by 2, 4 and 6 mass units, respectively. FIG. 3Bexemplifies a drug treatment of samples before standard is added. Afterpurification and optional quantification of each biomolecule, thesebiomolecules can be spiked in as standards.

Then MS, for example, MALDI, may be used to identify and compare orquantify amounts of each peptide and source protein from the samples.During MS analysis, the relative abundance between the heavy and lightisotope isoforms can be measured. Further, the mass difference betweenthe heavy and light isotope isoforms can be used to identify thespecific protein in the mixture that produced that proteolytic fragment.In this example, no further sequencing information is needed tocorroborate the identity of the mass ion.

Although this invention has been described in certain specificembodiments, many additional modifications and variations would beapparent to those skilled in the art. It is therefore to be understoodthat this invention may be practiced other than as specificallydescribed. Thus, the present embodiments of the invention should beconsidered in all respects as illustrative and not restrictive.

What is claimed is:
 1. A kit comprising at least one amino acidcontaining two or more atomic isotopes that are different in molecularmass from naturally-occurring isotopes, wherein the at least one aminoacid is not incorporated into a peptide or protein; and one or more of:a protein synthesis system; a template instruction for synthesizing abiomolecule standard; a resin for purifying said biomolecule standard; aprotein quantitation reagent; an enzyme or chemical reagent capable ofcleaving said biomolecule standard; an enzyme or chemical preparationcapable of cleaving said biomolecule standard; a vector; and apolymerase.
 2. The kit of claim 1, wherein said synthesis systemcomprises an extract selected from the group consisting of a bacterialextract, a eukaryotic extract, a plant extract, a mammalian extract, andan insect extract.
 3. The kit of claim 1, further comprising at leastone buffer for in vitro protein synthesis.
 4. The kit of claim 1,wherein at least one isotope of said two or more atomic isotopes is anatom selected from the group consisting of ¹⁵N, ¹³C, ¹⁸O, ²H and ³⁴S. 5.The kit of claim 1, wherein said enzyme is selected from the groupconsisting of trypsin, Endo-Lys-C, Endo-Glu-C, AspN protease, a yeastpeptidase, V-8 protease, pepsin, subtilisin, proteinase lc, tobacco etchvirus protease, and an enzyme with cleavage characteristics similar totrypsin.